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Project description:We constructed a small RNA cDNA library, using small RNA fraction with a length of 19-29 bases, and we performed deep sequencing of the cDNA library. R. conorii subsp. conorii strain Malish 7 was cultivated on XTC cells for 3 days at 28°C. RNA enriched with small RNA fractions was further extracted and cDNA was synthesized.

Project description:Rickettsia conorii are obligate intracellular bacteria responsible for the Mediterranean spotted fever. Their adaptation to highly divergent environments ranging from the arthropod vector to the human beings is essential to survival and virulence. Through a combination of total RNA purification and random prokaryotic cDNA amplification, we successfully analyzed transcription profiles by microarrays starting from 100 ng of R. conorii RNA. Real-time quantitative PCR results performed on unpurified total RNA was consistent with microarray measurements. Here, from the selected targets spotted on our microarray, we showed that in R. conorii, nutrient stress is accompanied by over-expression of the virB operon. Regulation of these genes could be mediated by the stringent response through ppGpp, consistent with the observed up-regulation of spoT and gmk. Exposure of R. conorii to a nutrient stress paradoxically reduced the expression of both GroEL and its transcriptional regulator RpoH. This shows that the over-expression of this chaperonin is a part of the natural life of intracellular growing rickettsiae. Our findings also evidenced that, unexpectedly, many atypical sequences, including small-size split genes, ORFans genes and highly conserved intergenic regions contains expressed sequences that are differentially regulated. The notable deficiency of transcriptional regulators within R. conorii genome does not reflect its incapability to undergo transcriptional changes but rather the existence of an alternative adaptation strategy. Keywords: Global transcriptional analysis Overall design: Rickettsia conorii were grown in confluent monolayers of African green monkey kidney cells (Vero cell, ATCC C1587). Combination of total RNA and random prokaryotic cDNA; measurements performed in triplicate.

Project description:We constructed a small RNA cDNA library, using small RNA fraction with a length of 19-29 bases, and we performed deep sequencing of the cDNA library. Overall design: R. conorii subsp. conorii strain Malish 7 was cultivated on XTC cells for 3 days at 28°C. RNA enriched with small RNA fractions was further extracted and cDNA was synthesized.

Project description:Rickettsia conorii, the causative agent of the MSF is transmitted to humans by the bite of infected ticks Rhipicephalus sanguineus. The skin thus constitutes an important barrier for the entry and propagation of R. conorii. Given this, analysis of the survival strategies used by the bacterium within infected skin is critical for our understanding of rickettsiosis. Here, we report the first genome-wide analysis of R. conorii gene expression from infected human skin biopsies. Our data showed that R. conorii exhibit a striking transcript signature that was remarkably conserved across patients, regardless of genotype. The expression profiles obtained using custom Agilent microarrays were validated by quantitative RT-PCR. Within eschars, approximately 15% (n = 211) of the total predicted R. conorii ORFs appeared differentially expressed. These genes are mostly down-regulated and encode proteins essential for bacterial replication. Some of the strategies displayed by rickettsiae to overcome the host defense barriers, thus avoiding killing, were also pointed out. The observed up-regulation of rickettsial genes associated with DNA repair is likely to correspond to a DNA-damaging agent enriched environment generated by the host cells to eradicate the pathogens. Survival of R. conorii within eschars also involves adaptation to osmotic stress, changes in cell surface proteins and up-regulation of some virulence factors. Interestingly, in contrast to down-regulated transcripts, we noticed that up-regulated ones rather exhibit a small nucleotide size, most of them being exclusive for the spotted fever group rickettsiae. On the overall, this work provides some insights on how R. conorii counteracts the host cell attack at the site of inoculation which corresponds to the front line of human host defense against rickettsia diffusion and that resembles to battlefield. 9 R.conorii clinical isolates from eschars co-hybridized with reference strain R.conorii Strain Malish 7

Project description:We used a microarray covering the whole genome of R. conorii to check if intergenic sequences were found transcribed. We checked the expression signals for probes corresponding to spacers as compared to probes corresponding to Open Reading Frames (ORFs). We got total RNA from R. conorii XTC cultures; we performed cDNA synthesis and then hybridizations. The hybridizations were repeated four times, and data were compared to check the reproducibility. Overall design: Bacterial culture. In this study, we used R. conorii subsp. conorii, strain Malish 7. It was cultivated on XTC cells for 3 days at 28°C. Microarray design. EArray (Agilent technologies) was used to generate a set of probes covering the whole genome of seven species of Rickettsiae (R. conorii strain Malish 7 (NC_003103), R. africae strain ESF-5 (NC_012633), R. slovaca (unpublished genome), R. massiliae (NC_009900), R. raoultii (unfinished genome), R. felis (NC_007109), R. prowazekii strain Madrid E (NC_000963)). Only CDSs >120 nt long and spacers >60 nt long were retained to generate probes. The probes are 60 nt long, following Agilent’s recommendations, except for those corresponding to 34 short predictions (see below) of 20 nt long. The probes present the following Tm properties: mean=78.82, minimum=66.34, maximum=85.16. Preceding steps yielded a final oligonucleotide set of 14,625 probes resulting to a final coverage of 96%. Microarray design included control spots. Expression microarrays and analysis. Using the previously obtained RNA, cDNA synthesis was performed with M-MLV reverse transcriptase (Invitrogen) following the manufacturer’s instructions. 1µg cDNA was labelled with Cy3-dCTP and Cy5-dCTP (GE Healthcare) using BioPrime DNA labelling System (Invitrogen). Labelled cDNA was purified using Minelute Reaction Cleanup kit (Qiagen). The levels of Cy3-dCTP and Cy5-dCTP incorporation were quantified by absorbance measurement at 550 nm and 650 nm, respectively, on Nanodrop (ThermoFisher). Hybridizations were performed for 17h at 62°C as previously described. Slides were scanned using Agilent scanner (Agilent Technologies, CA, USA) with a 3 µm resolution and a 20 bit format. Data were extracted by Feature Extraction TM software (10.5.1.1 version, Agilent). Data were then treated using MIDAS software (Microarray Data Analysis System) (http://www.tm4.org/midas.html) to perform a global normalization. Data were analyzed using Excel 2003 (Microsoft). A cut-off value defined as 2-standard deviation obtained for background intensities was then applied. The microarray data were analyzed after a global normalization. The hybridizations were repeated four times, and data were compared to check the reproducibility. The hybridization pattern after normalization showed a linear correlation in the relative gene expression; the correlation’s coefficients for the four repeated assays are presented in table 3A. They vary from 0.9468 to 0.9845 highlighting a good reproducibility of the experiments.